VEGFR-2Cell Membrane Chromatography For Screening Inhibitors

Backgrounds:The occurrence of cancer is a very complicated process, tumor angiogenesis not only convey nutrition for the growth of tumor cells, but also play an important role in invasion and metastasis of tumor growth, invasion and metastases and tumor angiogenesis are influenced by many factors. Vascular endothelial growth factor (VEGF) is a very important factor in the process of angiogenesis. It acts as a highly specific mitogen directly involving in the process of angiogenesis, which induces endothelial cells division and proliferation and also increases vascular permeability. VEGF performs its function through binding to special receptors. Three high-affinity cognate endothelia receptors for VEGF have been identified: VEGFR-1/Flt-1、VEGFR-2/KDR/Flk-1、VEGFR-3/Flt-4. These receptors belong to the subfamily of class III receptor tyrosine kinases (RTKs). Studies have indicated that KDR is the major signal transducer for the differentiation and proliferation of endothelial cells. VEGF binding to KDR, that results in dimerization of receptor monomers, transphosphorylation by dimerized receptors and docking of signaling proteins to receptor phosphotyrosines. An increase in the intrinsic catalytic activity and creation of binding sites on the RTKs to recruit cytoplasmic signaling proteins are primary features of RTKs activation, resulting in endothelial cells-associated reaction. As angiogenesis is of crucial importance for tumor growth, effectively inhibiting KDR signaling is considered as an attractive target for drugs screening novel anticancer agents against tumor angiogenesis.The screening methods of VEGFR-2inhibitors major on detecting its activity at present. But some orthodox methods have several shortcomings to be impossible avoided and are not fit for screening multicomponent and a great quantity of compounds, such as immobilized enzyme chromatography, immunizing analytical method and radioactive binding analysis method. At the same time, combinatorial chemistry technology accelerates synthesis speed of new drugs and leading compounds. So it is very necessary to develop a feasible and rapid screening platform.Owing to above reasons, cell membrane chromatography about VEGFR-2was developed in the thesis. And it has been applied for the preliminary screening of a batch of compounds.Methods:This experiment contrasted silica gel, chitosan/SiO2(CTS/SiO2) and sodium alginate/SiO2(ALG/SiO2) carrier material preparation. They are used to establish the cell membrane stationary phase VEGFR-2cell membrane chromatography.In this paper, two kinds of organic and inorganic hybrid materials were CTS/SiO2and ALG/SiO2had been preparation. First of all, the deacetylation degree of chitosan and the content of silicon were determinated. Because of silicon and large particle surface leads to fully epichlorohydrin reaction, the preparation of CTS/SiO2hybrid material use epichlorohydrin modified SiO2particle, which cross linked with chitosan. L18(37) orthogonal test was investigated in the amount of catalysts, reaction time and reaction temperature, the result is showed as chitosan content in hybrid material. In the presence of calcium ions, sodium alginate can make the alginate crosslinking gel. SiO2particles spread in sodium alginate solution, and the CaCl2solution drops into the formation of calcium alginate/SiO2gel. After curing formed and washed ALG/SiO2hybrid material, experiment inspected the proportion of sodium alginate and silica and the concentration of CaCl2solution. Scanning electron microscopy, infrared spectrum and thermogravimetric analysis and elemental analysis were used to evaluate two hybrid materials. Immobilized gelatinases methods were used to assess biocompatibility of different carrier, and calculation of the enzyme characteristic parameters-Km.The second important content of this work is high expression VEGFR-2cell membrane chromatography screening and cell membrane preparation. Cell surface VEGFR-2antigen of several tumor cells were determination by the flow cytometry. The result is that U251is the highest expression VEGFR-2. By differential centrifugal to get good active and purity of cell membranes, and inspected the cell membrane access of broken way, centrifugal speed, temperature on the cell membrane protein and the influence of the activity. Then cell membrane adsorption and fusion to the surface of carrier materials become cell membrane fixed phase. Then we determinate the ratio of the cell membrane and carrier factors to determine the optimal experiment project, and the influence of the amount of cell membrane protein and activity in different carrier.Screening inhibitors:Sunitinib to be listed for positive control is determined the retention time in VEGFR-2cell membrane chromatographic column, to verify the reliability of the models, and investigate the precision. Sample the new synthesis10compounds of anthranilic diamides, that is VEGFR-2small molecule inhibitors, and determine each compound’s retention time and capacity factor in the column. Comparing screening results of three kinds of chromatographic solid phase, and compare with the screening results of MTT method.Results:the best reaction conditions of CTS/SiO2are70℃reaction4h,0.1Og NaOH (catalyst). Scanning electron microscope (SEM) analysis results for chitosan cross linking group of silica particles. Infrared spectrum (IR) shows evident chitosan and silica characteristic peak. Thermogravimetric analysis (TGA) shows that the hybrid material thermal performance depression, and physical mixture with obvious difference. Element analysis was evident from the hybrid material in the quality of chitosan concentration was37.3%. ALG/SiO2hybrid material preparation process sodium alginate and the optimal proportion of silica are1:2. CaCl2solution of the optimal concentration of1.0mol/L, SEM for its shape irregular form silica embedding shuttle and calcium alginate mesh structure. The IR is also can see that sodium alginate and silica characteristic peak. TGA shows that the hybrid material thermal performance improved. Element analysis evident in the hybrid material sodium alginate quality score of30.9%. Comparison silica and two kinds of materials on the gelatinases the immobilized quantity and Km, immobilized enzyme quantity of silica is4848±80U/g,0.33±0.0015Km, CTS/SiO2immobilized enzyme amount to5094±59U/g,0.075±0.00018Km, ALG/SiO2immobilized enzyme amount to5388±21U/g,0.035±0.00019Km.Flow cytometric screening VEGFR-2is the highest expression of the human brain tumor cells is U251. Under4℃conditions, the cell ultrasound is the optimal way to broken cells, low speed600g centrifuged10min, again the14000g centrifuged25min to collect cell membranes. Then, the best proportion of cell membranes and carrier is2×106/0.01g.Cell membrane chromatography to determine the retention time of positive drug sunitinib is the longest, and the stability and repeatability are good, MTT method also can evident that its IC50is760nM, so it shows that screening model is feasible. To10other compounds, the three kinds of cell membrane chromatography and MTT method to determine the overall results keep consistent. The difference among all the compounds that combination activity with cell membrane is large, but in general drugs in silica cell membrane on the chromatographic retention time is slightly long, CTS/SiO2cell membrane chromatography screening performance is the best.Conclusion:The screening methods developed in the thesis are accurate and feasible. The rapid screen of multi-component and a great quantity of compounds can come true.